Highly sensitive detection of a HER2 12-base pair duplicated insertion mutation in lung cancer using the Eprobe-PCR method

• Published in: PloS one 2017-02, Vol.12 (2), p.e0171225-e0171225
• Main Authors: Takase, Yoshiaki, Usui, Kengo, Shimizu, Kimihiro, Kimura, Yasumasa, Ichihara, Tatsuo, Ohkawa, Takahiro, Atsumi, Jun, Enokida, Yasuaki, Nakazawa, Seshiru, Obayashi, Kai, Ohtaki, Yoichi, Nagashima, Toshiteru, Mitani, Yasumasa, Takeyoshi, Izumi
• Format: Article
• Language: English
• Subjects: Adult; Aged; Aged, 80 and over; Analysis; Antibody diversity; Bioinformatics; Biology and Life Sciences; Cancer; Cancer therapies; Deoxyribonucleic acid; Design; Diagnosis; Diagnostic systems; Dilution; DNA; DNA sequencing; Epidermal growth factor; Epidermal growth factors; ErbB-2 protein; Female; Gene amplification; Genes, erbB-2 - genetics; Genetic aspects; Health aspects; High-Throughput Nucleotide Sequencing - methods; Humans; Hybridization; Insertion; Kinases; Life sciences; Lung cancer; Lung diseases; Lung Neoplasms - genetics; Male; Medical prognosis; Medicine; Medicine and Health Sciences; Methods; Middle Aged; Mutagenesis, Insertional - genetics; Mutation; Paraffin; Plasmids; Polymerase chain reaction; Polymerase Chain Reaction - methods; Reproducibility of Results; Reproduction (copying); Research and Analysis Methods; Sensitivity; Sensitivity and Specificity; Surgery; Tissues; University graduates
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0171225

Somatic mutation in human epidermal growth factor receptor-related 2 gene (HER2) is one of the driver mutations in lung cancer. HER2 mutations are found in about 2% of lung adenocarcinomas (ADCs). Previous reports have been based mainly on diagnostic screening by Sanger sequencing or next-generation sequencing (NGS); however, these methods are time-consuming and complicated. We developed a rapid, simple, sensitive mutation detection assay for detecting HER2 12 base pair-duplicated insertion mutation based on the Eprobe-mediated PCR method (Eprobe-PCR) and validated the sensitivity of this assay system for clinical diagnostics. We examined 635 tumor samples and analyzed HER2 mutations using the Eprobe-PCR method, NGS, and Sanger sequencing. In a serial dilution study, the Eprobe-PCR was able to detect mutant plasmid DNA when its concentration was reduced to 0.1% by mixing with wild-type DNA. We also confirmed amplification of the mutated plasmid DNA with only 10 copies per reaction. In ADCs, Eprobe-PCR detected the HER2 mutation in 2.02% (9/446), while Sanger sequencing detected it in 1.57% (7/446). Eprobe-PCR was able to detect the mutation in two samples that were undetectable by Sanger sequencing. All non-ADC samples were wild-type. There were no discrepancies between frozen and formalin-fixed paraffin-embedded tissues in the nine samples. HER2 mutations detected by NGS data validated the high sensitivity of the method. Therefore, this new technique can lead to precise molecular-targeted therapies.